This invention relates to a method and apparatus for injecting sample into a capillary electrophoresis process from a membrane.
Capillary electrophoresis (CE) is an efficient analytical separation technique for analysis of minute amounts of sample. CE separations are performed in a narrow diameter capillary tube, which is filled with an electrically conductive medium termed the "carrier electrolyte." An electric field is applied between the two ends of the capillary tube, and species in the sample move from one electrode toward the other electrode at a rate which is dependent on the electrophoretic mobility of each species as well as on the rate of fluid movement in the tube. CE may be performed using gels or liquids, such as buffers, in the capillary. In one liquid mode, known as free zone electrophoresis, separations are based on differences in the free solution mobility of sample species. In another liquid mode, micelles are used to effect separations based on differences in hydrophobicity. This is known as Micellar Electrokinetic Capillary Chromatography (MECC). In capillary gel electrophoresis, an electrically conductive gel fills the capillary tube rather than the liquid electrolyte. The gel function as an anticonnective support to minimize sample band spreading. In free zone electrophoresis, a high moleular weight solute such as polyethylene oxide or hydroxymethylcellulose can be added to the solvent to provide anticonvective effects analagous to that supplied by the gel.
CE is advantageous for several reasons. These include fast separation speed, high resolution and small sample size. For example, separation speeds using CE can be 10 to 20 times faster than conventional gel electrophoresis, and no post-run staining is necessary. In part, high resolution can be obtained through the use of high voltages because of the rapid dissipation of heat by the capillary. Further, band broadening is minimized due to the narrow capillary inner diameter. In electrophoresis, the phenomenon of electroosmosis, or electroosmotic flow (EOF) occurs. This is a bulk flow of liquid which affects all of the sample molecules regardless of charge. EOF can contribute to improved resolution or separation speed in free-zone CE.
In order to achieve the high resolution that CE is capable of, it is necessary that the sample be confined to a narrow starting zone when the electrophoretic process begins. This limits the volume of sample that can be introduced into the capillary to a very small fraction of the total capillary volume. In CE, only a few nanoliters or less of the sample solution is introduced into the capillary. Injection volumes that are much larger than this volume degrade the resolution of the CE method. In capillary electrophoresis as generally practiced today, sample solutions to be analyzed are introduced from vials. The vials must contain at least a few microliters of sample solution in order that the capillary tip can be immersed in the sample solution. After sample introduction, the capillary tip is immersed in electrolyte solution and a voltage is applied across both ends of the capillary to perform the electrophoretic separation.
The disparity described above between the volume of sample required for the physical mechanism of sample introduction (at least a few microliters) and the volume actually introduced (a few nanoliters or less) is a disadvantage of current systems. Also, in some cases samples are not readily available in solution form, such as when the sample stems from a gel electrophoresis separation and exists as a spot or band on a blotting membrane.
It would be desirable to provide a means for introducing samples into a capillary electrophoresis apparatus which does not require a large sample volume. In addition, it would be desirable to provide a means for introducing sample from sample carriers other than a solvent. It would be desirable to introduce into the capillary a larger percentage of the total available sample volume than can be effected with presently available injection procedures.